RESUMO
It is widely acknowledged that immunoglobulins (Igs) are produced solely by B-lineage cells. The Ig gene is created by the rearrangement of a group of gene segments [variable (V), diversity (D), and joining (J) segments rearrangement, or V(D)J recombination], which results in the vast diversity of B cell-derived Ig responsible for recognising various antigens. Ig subsequently undergoes somatic hypermutation (SHM) and class switch recombination (CSR) after exposure to antigens, thus converting the low-affinity IgM to IgG, IgA, or IgE antibodies. IgM and IgD are primarily expressed in naïve B cells that have not been exposed to antigens, they do not undergo somatic hypermutation; hence, their variable region sequences remain the same as those in the germline. In contrast, IgG, IgA, and IgE are expressed in antigen-stimulated memory B cells or plasma cells, and thus, they often possess high-frequency mutations in their variable region sequences. Since the discovery that Ig can be produced by non-B cells, Qiu's group has investigated and compared the genetic characteristics of B cell-derived Ig and non-B cell-derived Ig. These findings demonstrated that non-B cell-derived Ig shares certain similarities with B cell-derived Ig in that the sequence of its constant region is identical to that of B cell-derived Ig, and its variable region is also strictly dependent on the rearrangement of V, D, and J gene segments. Moreover, akin to B cell-derived Ig, the V regions of IgM and IgD are rarely mutated, while IgG, IgA, and IgE produced by cancer cells are frequently mutated. However, the non-B cell-derived Ig V region sequence displays unique characteristics. (1) Unlike the vast diversity of B cell-derived Igs, non-B cell-derived Igs exhibit restricted diversity; cells from the same lineage always select the same V(D)J recombination patterns; (2) Both mRNA and proteins of RAG1/RAG2 recombinase have been detected in Ig positive cancer cell lines and normal tissues. But Ig recombination could also be found in RAG1-/- and RAG2-/- mice, suggesting that they are not necessary for the rearrangement of non-B cell-derived Igs. These features of non-B cell-derived Igs suggest a potentially undiscovered mechanism of V(D)J recombination, ligation, and SHM in non-B cells, which necessitates further investigation with advanced technology in molecular biology.
Assuntos
Linfócitos B , Genes de Imunoglobulinas , Animais , Humanos , Camundongos , Linfócitos B/imunologia , Genes de Imunoglobulinas/genética , Switching de Imunoglobulina/genética , Switching de Imunoglobulina/imunologia , Imunoglobulinas/genética , Imunoglobulinas/imunologia , Hipermutação Somática de Imunoglobulina/genéticaRESUMO
Division tracking dyes like Cell Trace Violet (CTV) enable the quantification of cell proliferation, division, and survival kinetics of human naïve B cell responses in vitro. Human naïve B cells exhibit distinct responses to different stimuli, with CpG and anti-Ig inducing a T cell-independent (TI) response, while CD40L and IL-21 promote a T cell-dependent (TD) response that induces isotype switching and differentiation into antibody-secreting cells (ASCs). Both stimulation methods yield valuable insights into the intrinsic programming of B cell health within individuals, making them useful for clinical investigations. For instance, quantitative analysis from these B cell populations could reveal biologically meaningful measurements such as the average number of division rounds and the time to cells' fate. Here, we describe a novel in vitro culture setup for CTV-labelled human naïve B cells and a method for obtaining precise time-based data on proliferation, division-linked isotype switching, and differentiation.
Assuntos
Linfócitos B , Técnicas de Cultura de Células , Diferenciação Celular , Proliferação de Células , Humanos , Linfócitos B/citologia , Linfócitos B/metabolismo , Técnicas de Cultura de Células/métodos , Cinética , Ativação Linfocitária , Células Cultivadas , Switching de ImunoglobulinaRESUMO
B cells and the antibodies they produce are critical in host defense against pathogens and contribute to various immune-mediated diseases. B cells responding to activating signals in vitro release extracellular vesicles (EV) that carry surface antibodies, yet B cell production of EVs that express antibodies and their function in vivo is incompletely understood. Using transgenic mice expressing the Cre recombinase in B cells switching to IgG1 to induce expression of fusion proteins between emerald green fluorescent protein (emGFP) and the EV tetraspanin CD63 as a model, we identify emGFP expression in B cells responding to foreign antigen in vivo and characterize the emGFP+ EVs they release. Our data suggests that emGFP+ germinal center B cells undergoing immunoglobulin class switching to express IgG and their progeny memory B cells and plasma cells, also emGFP+, are sources of circulating antigen-specific IgG+ EVs. Furthermore, using a mouse model of influenza virus infection, we find that IgG+ EVs specific for the influenza hemagglutinin antigen protect against virus infection. In addition, crossing the B cell Cre driver EV reporter mice onto the Nba2 lupus-prone strain revealed increased circulating emGFP+ EVs that expressed surface IgG against nuclear antigens linked to autoimmunity. These data identify EVs loaded with antibodies as a novel route for antibody secretion in B cells that contribute to adaptive immune responses, with important implications for different functions of IgG+ EVs in infection and autoimmunity.
Assuntos
Linfócitos B , Vesículas Extracelulares , Imunoglobulina G , Camundongos Transgênicos , Animais , Vesículas Extracelulares/imunologia , Vesículas Extracelulares/metabolismo , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Camundongos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Infecções por Orthomyxoviridae/imunologia , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genética , Antígenos/imunologia , Switching de Imunoglobulina , Camundongos Endogâmicos C57BL , Centro Germinativo/imunologia , Centro Germinativo/metabolismoRESUMO
Previous studies have demonstrated the efficacy and feasibility of an anti-viral vaccine strategy that takes advantage of pre-existing CD4+ helper T (Th) cells induced by Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccination. This strategy uses immunization with recombinant fusion proteins comprised of a cell surface expressed viral antigen, such as a viral envelope glycoprotein, engineered to contain well-defined BCG Th cell epitopes, thus rapidly recruiting Th cells induced by prior BCG vaccination to provide intrastructural help to virus-specific B cells. In the current study, we show that Th cells induced by BCG were localized predominantly outside of germinal centers and promoted antibody class switching to isotypes characterized by strong Fc receptor interactions and effector functions. Furthermore, BCG vaccination also upregulated FcγR expression to potentially maximize antibody-dependent effector activities. Using a mouse model of Ebola virus (EBOV) infection, this vaccine strategy provided sustained antibody levels with strong IgG2c bias and protection against lethal challenge. This general approach can be easily adapted to other viruses, and may be a rapid and effective method of immunization against emerging pandemics in populations that routinely receive BCG vaccination.
Assuntos
Anticorpos Antivirais , Vacina BCG , Vacinas contra Ebola , Ebolavirus , Doença pelo Vírus Ebola , Animais , Camundongos , Vacina BCG/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Doença pelo Vírus Ebola/imunologia , Vacinas contra Ebola/imunologia , Vacinas contra Ebola/administração & dosagem , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Linfócitos T Auxiliares-Indutores/imunologia , Vacinação/métodos , Camundongos Endogâmicos C57BL , Feminino , Humanos , Modelos Animais de Doenças , Receptores de IgG/imunologia , Desenvolvimento de Vacinas , Switching de Imunoglobulina , ImunizaçãoRESUMO
BACKGROUND: RNA-DNA hybrids or R-loops are associated with deleterious genomic instability and protective immunoglobulin class switch recombination (CSR). However, the underlying phenomenon regulating the two contrasting functions of R-loops is unknown. Notably, the underlying mechanism that protects R-loops from classic RNase H-mediated digestion thereby promoting persistence of CSR-associated R-loops during CSR remains elusive. RESULTS: Here, we report that during CSR, R-loops formed at the immunoglobulin heavy (IgH) chain are modified by ribose 2'-O-methylation (2'-OMe). Moreover, we find that 2'-O-methyltransferase fibrillarin (FBL) interacts with activation-induced cytidine deaminase (AID) associated snoRNA aSNORD1C to facilitate the 2'-OMe. Moreover, deleting AID C-terminal tail impairs its association with aSNORD1C and FBL. Disrupting FBL, AID or aSNORD1C expression severely impairs 2'-OMe, R-loop stability and CSR. Surprisingly, FBL, AID's interaction partner and aSNORD1C promoted AID targeting to the IgH locus. CONCLUSION: Taken together, our results suggest that 2'-OMe stabilizes IgH-associated R-loops to enable productive CSR. These results would shed light on AID-mediated CSR and explain the mechanism of R-loop-associated genomic instability.
Assuntos
Citidina Desaminase , Switching de Imunoglobulina , Estruturas R-Loop , Switching de Imunoglobulina/genética , Citidina Desaminase/metabolismo , Citidina Desaminase/genética , Citidina Desaminase/química , Animais , Camundongos , Metilação , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Recombinação Genética , RNA/metabolismo , RNA/genéticaRESUMO
Humanized mice are limited in terms of modeling human immunity, particularly with regards to antibody responses. Here we constructed a humanized (THX) mouse by grafting non-γ-irradiated, genetically myeloablated KitW-41J mutant immunodeficient pups with human cord blood CD34+ cells, followed by 17ß-estradiol conditioning to promote immune cell differentiation. THX mice reconstitute a human lymphoid and myeloid immune system, including marginal zone B cells, germinal center B cells, follicular helper T cells and neutrophils, and develop well-formed lymph nodes and intestinal lymphoid tissue, including Peyer's patches, and human thymic epithelial cells. These mice have diverse human B cell and T cell antigen receptor repertoires and can mount mature T cell-dependent and T cell-independent antibody responses, entailing somatic hypermutation, class-switch recombination, and plasma cell and memory B cell differentiation. Upon flagellin or a Pfizer-BioNTech coronavirus disease 2019 (COVID-19) mRNA vaccination, THX mice mount neutralizing antibody responses to Salmonella or severe acute respiratory syndrome coronavirus 2 Spike S1 receptor-binding domain, with blood incretion of human cytokines, including APRIL, BAFF, TGF-ß, IL-4 and IFN-γ, all at physiological levels. These mice can also develop lupus autoimmunity after pristane injection. By leveraging estrogen activity to support human immune cell differentiation and maturation of antibody responses, THX mice provide a platform to study the human immune system and to develop human vaccines and therapeutics.
Assuntos
Anticorpos Neutralizantes , Switching de Imunoglobulina , Animais , Humanos , Camundongos , Anticorpos Neutralizantes/imunologia , Linfócitos B/imunologia , SARS-CoV-2/imunologia , COVID-19/imunologia , Anticorpos Antivirais/imunologia , Hipermutação Somática de Imunoglobulina , Diferenciação Celular/imunologiaRESUMO
Humoral immunity depends on the germinal center (GC) reaction where B cells are tightly controlled for class-switch recombination and somatic hypermutation and finally generated into plasma and memory B cells. However, how protein SUMOylation regulates the process of the GC reaction remains largely unknown. Here, we show that the expression of SUMO-specific protease 1 (SENP1) is up-regulated in GC B cells. Selective ablation of SENP1 in GC B cells results in impaired GC dark and light zone organization and reduced IgG1-switched GC B cells, leading to diminished production of class-switched antibodies with high-affinity in response to a TD antigen challenge. Mechanistically, SENP1 directly binds to Paired box protein 5 (PAX5) to mediate PAX5 deSUMOylation, sustaining PAX5 protein stability to promote the transcription of activation-induced cytidine deaminase. In summary, our study uncovers SUMOylation as an important posttranslational mechanism regulating GC B cell response.
Assuntos
Linfócitos B , Cisteína Endopeptidases , Centro Germinativo , Fator de Transcrição PAX5 , Sumoilação , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Fator de Transcrição PAX5/metabolismo , Fator de Transcrição PAX5/genética , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/genética , Camundongos , Switching de Imunoglobulina , Humanos , Citidina Desaminase/metabolismo , Citidina Desaminase/genética , Imunidade Humoral , Camundongos Endogâmicos C57BLRESUMO
Activation induced cytidine deaminase (AID) is a key element of the adaptive immune system, required for immunoglobulin isotype switching and affinity maturation of B-cells as they undergo the germinal center (GC) reaction in peripheral lymphoid tissue. The inherent DNA damaging activity of this enzyme can also have off-target effects in B-cells, producing lymphomagenic chromosomal translocations that are characteristic features of various classes of non-Hodgkin B-cell lymphoma (B-NHL), and generating oncogenic mutations, so-called aberrant somatic hypermutation (aSHM). Additionally, AID has been found to affect gene expression through demethylation as well as altered interactions between gene regulatory elements. These changes have been most thoroughly studied in B-NHL arising from GC B-cells. Here, we describe the most common classes of GC-derived B-NHL and explore the consequences of on- and off-target AID activity in B and plasma cell neoplasms. The relationships between AID expression, including effects of infection and other exposures/agents, mutagenic activity and lymphoma biology are also discussed.
Assuntos
Linfócitos B , Citidina Desaminase , Centro Germinativo , Linfoma de Células B , Humanos , Citidina Desaminase/metabolismo , Citidina Desaminase/genética , Centro Germinativo/imunologia , Linfoma de Células B/genética , Linfoma de Células B/imunologia , Animais , Linfócitos B/imunologia , Hipermutação Somática de Imunoglobulina , Regulação Neoplásica da Expressão Gênica , Switching de ImunoglobulinaRESUMO
Statistical and machine learning methods have proved useful in many areas of immunology. In this paper, we address for the first time the problem of predicting the occurrence of class switch recombination (CSR) in B-cells, a problem of interest in understanding antibody response under immunological challenges. We propose a framework to analyze antibody repertoire data, based on clonal (CG) group representation in a way that allows us to predict CSR events using CG level features as input. We assess and compare the performance of several predicting models (logistic regression, LASSO logistic regression, random forest, and support vector machine) in carrying out this task. The proposed approach can obtain an unweighted average recall of 71 % $71\%$ with models based on variable region descriptors and measures of CG diversity during an immune challenge and, most notably, before an immune challenge.
Assuntos
Linfócitos B , Switching de Imunoglobulina , Linfócitos B/imunologia , Animais , Biometria/métodos , Recombinação Genética , Anticorpos/imunologia , Camundongos , HumanosRESUMO
Immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome, a rare autosomal recessive disorder, manifests with hypoglobulinemia and chromosomal instability accompanied by DNA hypomethylation. Pathological variants in the DNMT3B, ZBTB24, CDCA7, or HELLS genes underlie its etiology. Activated lymphocytes from patients often display distinctive multiradial chromosomes fused via pericentromeric regions. Recent studies have provided deeper insights into how pathological variants in ICF-related proteins cause DNA hypomethylation and chromosome instability. However, the understanding of the molecular pathogenesis underlying immunodeficiency is still in its nascent stages. In the past half-decade, the roles of CDCA7, HELLS, and ZBTB24 in classical non-homologous end joining during double-strand DNA break repair and immunoglobulin class-switch recombination (CSR) have been unveiled. Nevertheless, given the decreased all classes of immunoglobulins in most patients, CSR deficiency alone cannot fully account for the immunodeficiency. The latest finding showing dysregulation of immunoglobulin signaling may provide a clue to understanding the immunodeficiency mechanism. While less common, a subgroup of patients exhibits T-cell abnormalities alongside B-cell anomalies, including reduced regulatory T-cells and increased effector memory T- and follicular helper T-cells. The dysregulation of immunoglobulin signaling in B-cells, the imbalance in T-cell subsets, and/or satellite RNA-mediated activation of innate immune response potentially explain autoimmune manifestations in a subset of patients. These findings emphasize the pivotal roles of ICF-related proteins in both B- and T-cell functions. ICF syndrome studies have illuminated many fundamental mechanisms. Further investigations will certainly continue to unveil additional mechanisms and their interplay.
Assuntos
Reparo do DNA , Epigênese Genética , Síndromes de Imunodeficiência , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Metilação de DNA , Animais , Switching de Imunoglobulina/genética , Doenças da Imunodeficiência Primária/genética , Doenças da Imunodeficiência Primária/imunologia , Doenças da Imunodeficiência Primária/diagnósticoRESUMO
IgE is induced by the presence of IL-4 by class switching from IgM through IgG1 to IgE. IL-21 inhibits the IgE class switch by induction of Blimp1 leading to Stat6 and AID downregulation, and plasmablast/plasma cell differentiation.
Assuntos
Diferenciação Celular , Switching de Imunoglobulina , Imunoglobulina E , Imunoglobulina G , Interleucinas , Plasmócitos , Fator 1 de Ligação ao Domínio I Regulador Positivo , Fator 1 de Ligação ao Domínio I Regulador Positivo/genética , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , Plasmócitos/imunologia , Diferenciação Celular/imunologia , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Animais , Interleucinas/metabolismo , Interleucinas/imunologia , Camundongos , Interleucina-4/imunologia , Interleucina-4/metabolismoRESUMO
Class switch recombination (CSR) diversifies the effector functions of antibodies and involves complex regulation of transcription and DNA damage repair. Here, we show that the deubiquitinase USP7 promotes CSR to immunoglobulin A (IgA) and suppresses unscheduled IgG switching in mature B cells independent of its role in DNA damage repair, but through modulating switch region germline transcription. USP7 depletion impairs Sα transcription, leading to abnormal activation of Sγ germline transcription and increased interaction with the CSR center via loop extrusion for unscheduled IgG switching. Rescue of Sα transcription by transforming growth factor ß (TGF-ß) in USP7-deleted cells suppresses Sγ germline transcription and prevents loop extrusion toward IgG CSR. Mechanistically, USP7 protects transcription factor RUNX3 from ubiquitination-mediated degradation to promote Sα germline transcription. Our study provides evidence for active transcription serving as an anchor to impede loop extrusion and reveals a functional interplay between USP7 and TGF-ß signaling in promoting RUNX3 expression for efficient IgA CSR.
Assuntos
Subunidade alfa 3 de Fator de Ligação ao Core , Imunoglobulina A , Switching de Imunoglobulina , Ativação Transcricional , Peptidase 7 Específica de Ubiquitina , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Animais , Imunoglobulina A/metabolismo , Peptidase 7 Específica de Ubiquitina/metabolismo , Peptidase 7 Específica de Ubiquitina/genética , Camundongos , Fator de Crescimento Transformador beta/metabolismo , Camundongos Endogâmicos C57BL , Humanos , Ubiquitinação , Linfócitos B/metabolismo , Linfócitos B/imunologia , Imunoglobulina G/metabolismo , Imunoglobulina G/imunologia , Estabilidade ProteicaRESUMO
Efficient repair of DNA double-strand breaks in the Ig heavy chain gene locus is crucial for B-cell antibody class switch recombination (CSR). The regulatory dynamics of the repair pathway direct CSR preferentially through nonhomologous end joining (NHEJ) over alternative end joining (AEJ). Here, we demonstrate that the histone acetyl reader BRD2 suppresses AEJ and aberrant recombination as well as random genomic sequence capture at the CSR junctions. BRD2 deficiency impairs switch (S) region synapse, optimal DNA damage response (DDR), and increases DNA break end resection. Unlike BRD4, a similar bromodomain protein involved in NHEJ and CSR, BRD2 loss does not elevate RPA phosphorylation and R-loop formation in the S region. As BRD2 stabilizes the cohesion loader protein NIPBL in the S regions, the loss of BRD2 or NIPBL shows comparable deregulation of S-S synapsis, DDR, and DNA repair pathway choice during CSR. This finding extends beyond CSR, as NIPBL and BRD4 have been linked to Cornelia de Lange syndrome, a developmental disorder exhibiting defective NHEJ and Ig isotype switching. The interplay between these proteins sheds light on the intricate mechanisms governing DNA repair and immune system functionality.
Assuntos
Proteínas que Contêm Bromodomínio , Reparo do DNA por Junção de Extremidades , Switching de Imunoglobulina , Fatores de Transcrição , Animais , Humanos , Camundongos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Proteínas que Contêm Bromodomínio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA , Switching de Imunoglobulina/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Recombinação Genética , Fatores de Transcrição/metabolismoRESUMO
We describe two types of IGH::BCL2 breakpoints involving the 5' region of BCL2 (5' BCL2). One was ins(14;18)(q32;q21q21) observed in 2 follicular lymphoma (FL) cases, in which IGH was cleaved at 3' of IGHD and 5' of IGHJ and BCL2 was cleaved at 5' BCL2 and downstream regions, and a 281- or 201-kilobase pair fragment containing the BCL2 protein-coding sequences was invertedly inserted into IGH. In another type observed in 2 FL and 2 chronic lymphocytic leukemia (CLL) cases, breakage and reunion occurred within the switch region associated with IGHM (Sµ) and 5' BCL2, creating IGH Sµ::5' BCL2 fusion sequences on der(18)t(14;18)(q32;q21). The former is considered to be mediated by VDJ-recombination, while the latter by the class switch recombination process. There were no particular features in FL or CLL cases with IGH::5' BCL2 breakpoints compared with those with t(14;18)(q32;q21)/IGH::BCL2 involving the 3' breakpoint cluster regions.
Assuntos
Pontos de Quebra do Cromossomo , Cromossomos Humanos Par 14 , Cromossomos Humanos Par 18 , Cadeias Pesadas de Imunoglobulinas , Proteínas Proto-Oncogênicas c-bcl-2 , Translocação Genética , Humanos , Cromossomos Humanos Par 14/genética , Cromossomos Humanos Par 18/genética , Cadeias Pesadas de Imunoglobulinas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Switching de Imunoglobulina/genética , Recombinação V(D)J/genética , Linfoma Folicular/genética , Linfoma Folicular/patologia , Pessoa de Meia-Idade , Masculino , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/patologia , Feminino , Hibridização in Situ Fluorescente , IdosoRESUMO
Transcription factors BACH2 and IRF4 are both essential for antibody class-switch recombination (CSR) in activated B lymphocytes, while they oppositely regulate the differentiation of plasma cells (PCs). Here, we investigated how BACH2 and IRF4 interact during CSR and plasma-cell differentiation. We found that BACH2 organizes heterochromatin formation of target gene loci in mouse splenic B cells, including targets of IRF4 activation such as Aicda, an inducer of CSR, and Prdm1, a master plasma-cell regulator. Release of these gene loci from heterochromatin in response to B-cell receptor stimulation was coupled to AKT-mTOR pathway activation. In Bach2-deficient B cells, PC genes' activation depended on IRF4 protein accumulation, without an increase in Irf4 mRNA. Mechanistically, a PU.1-IRF4 heterodimer in activated B cells promoted BACH2 function by inducing gene expression of Bach2 and Pten, a negative regulator of AKT signaling. Elevated AKT activity in Bach2-deficient B cells resulted in IRF4 protein accumulation. Thus, BACH2 and IRF4 mutually modulate the activity of each other, and BACH2 inhibits PC differentiation by both the repression of PC genes and the restriction of IRF4 protein accumulation.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica , Diferenciação Celular , Fatores Reguladores de Interferon , Plasmócitos , Animais , Camundongos , Linfócitos B/metabolismo , Linfócitos B/imunologia , Linfócitos B/citologia , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Diferenciação Celular/genética , Heterocromatina/metabolismo , Heterocromatina/genética , Switching de Imunoglobulina/genética , Fatores Reguladores de Interferon/metabolismo , Fatores Reguladores de Interferon/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Plasmócitos/metabolismo , Plasmócitos/imunologia , Plasmócitos/citologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , PTEN Fosfo-Hidrolase/genética , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/genética , Transativadores/metabolismo , Transativadores/genéticaRESUMO
Memory B cells (MBCs) play a critical role in protection against homologous and variant pathogen challenge by either differentiating to plasma cells (PCs) or to germinal center (GC) B cells. The human MBC compartment contains both switched IgG+ and unswitched IgM+ MBCs; however, whether these MBC subpopulations are equivalent in their response to B cell receptor cross-linking and their resulting fates is incompletely understood. Here, we show that IgG+ and IgM+ MBCs can be distinguished based on their response to κ-specific monoclonal antibodies of differing affinities. IgG+ MBCs responded only to high-affinity anti-κ and differentiated almost exclusively toward PC fates. In contrast, IgM+ MBCs were eliminated by apoptosis by high-affinity anti-κ but responded to low-affinity anti-κ by differentiating toward GC B cell fates. These results suggest that IgG+ and IgM+ MBCs may play distinct yet complementary roles in response to pathogen challenge ensuring the immediate production of high-affinity antibodies to homologous and closely related challenges and the generation of variant-specific MBCs through GC reactions.
Assuntos
Switching de Imunoglobulina , Células B de Memória , Humanos , Linfócitos B , Antígenos , Imunoglobulina G , Imunoglobulina M , Memória ImunológicaRESUMO
Class switch recombination (CSR) plays an important role in adaptive immune response by enabling mature B cells to replace the initial IgM by another antibody class (IgG, IgE or IgA). CSR is preceded by transcription of the IgH constant genes and is controlled by the super-enhancer 3' regulatory region (3'RR) in an activation-specific manner. The 3'RR is composed of four enhancers (hs3a, hs1-2, hs3b and hs4). In mature B cells, 3'RR activity correlates with transcription of its enhancers. CSR can also occur in primary developing B cells though at low frequency, but in contrast to mature B cells, the transcriptional elements that regulate the process in developing B cells are ill-known. In particular, the role of the 3'RR in the control of constant genes' transcription and CSR has not been addressed. Here, by using a mouse line devoid of the 3'RR and a culture system that highly enriches in pro-B cells, we show that the 3'RR activity is indeed required for switch transcription and CSR, though its effect varies in an isotype-specific manner and correlates with transcription of hs4 enhancer only.
Assuntos
Cadeias Pesadas de Imunoglobulinas , Super Intensificadores , Cadeias Pesadas de Imunoglobulinas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Switching de Imunoglobulina/genética , Linfócitos B , Isotipos de Imunoglobulinas/genética , Elementos Facilitadores GenéticosRESUMO
BACKGROUND: The noninflammatory immunoglobulin G4 (IgG4) is linked to tolerance and is unique to humans. Although poorly understood, prolonged antigenic stimulation and IL-4-signaling along the T helper 2-axis may be instrumental in IgG4 class switching. Recently, repeated SARS-CoV-2 mRNA vaccination has been linked to IgG4 skewing. Although widely used immunosuppressive drugs have been shown to only moderately affect humoral responses to SARS-CoV-2 mRNA vaccination, the effect on IgG4 switching has not been investigated. METHODS: Here we study the impact of such immunosuppressive drugs, including the IL-4 receptor-blocking antibody dupilumab, on IgG4 skewing upon repeated SARS-CoV-2 mRNA vaccination. Receptor-binding domain (RBD) specific antibody responses were longitudinally measured in 600 individuals, including patients with immune-mediated inflammatory diseases treated with a TNF inhibitor (TNFi) and/or methotrexate (MTX), dupilumab, and healthy/untreated controls, after repeated mRNA vaccination. RESULTS: We observed a substantial increase in the proportion of RBD-specific IgG4 antibodies (median 21%) in healthy/untreated controls after third vaccination. This IgG4 skewing was profoundly reduced in dupilumab-treated patients (<1%). Unexpectedly, an equally strong suppression of IgG4 skewing was observed in TNFi-treated patients (<1%), whereas MTX caused a modest reduction (7%). RBD-specific total IgG levels were hardly affected by these immunosuppressive drugs. Minimal skewing was observed, when primary vaccination was adenoviral vector-based. CONCLUSIONS: Our results imply a critical role for IL-4/IL-13 as well as TNF in vivo IgG4 class switching. These novel findings advance our understanding of IgG4 class switch dynamics, and may benefit humoral tolerance induction strategies, treatment of IgG4 pathologies and mRNA vaccine optimization.
Assuntos
Anticorpos Monoclonais Humanizados , Switching de Imunoglobulina , Imunoglobulina G , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/sangue , Feminino , Masculino , Pessoa de Meia-Idade , SARS-CoV-2/imunologia , Inibidores do Fator de Necrose Tumoral/uso terapêutico , COVID-19/prevenção & controle , COVID-19/imunologia , Adulto , Vacinas de mRNA/imunologia , Idoso , Vacinação , Vacinas contra COVID-19/imunologia , Imunossupressores/uso terapêutico , Imunossupressores/farmacologia , Anticorpos Antivirais/imunologiaRESUMO
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant and high affinity ligand for the aryl hydrocarbon receptor (AhR). In animal models, AhR activation by TCDD generally inhibits antibody secretion. However, it is less clear if this translates to human antibody production. Using a human Burkitt lymphoma B-cell line (CL-01) that can be stimulated to secrete Ig and undergo class switch recombination to other Ig isotypes, the current study evaluated the effects of AhR activation or antagonism on the human Ig isotypic expression profile with CD40L+IL-4 stimulation. Our results suggest that AhR agonists (TCDD and indirubin) have little to no effect on IgM or IgA secretion, which were also not induced with stimulation. However, AhR activation significantly inhibited stimulation-induced IgG secretion, an effect reversed by the AhR antagonist CH223191. Evaluation of Ig heavy chain (IgH) constant region gene expression (ie Cµ, Cγ1-4, Cα1-2, and Cε that encode for IgM, IgG1-4, IgA1-2, and IgE, respectively) demonstrated differential effects. While Cµ and Cα2 transcripts were unaffected by stimulation or AhR agonists, AhR activation significantly inhibited stimulation-induced Cγ2-4 and Cε mRNA transcripts, which was reversed by AhR antagonism. Notably, AhR antagonism in the absence of exogenous AhR ligands significantly increased IgG and IgA secretion as well as the expression of Cγ2-4 and Cε. These results suggest that modulation of AhR activity differentially alters the IgH isotypic expression profile and antibody secretion that may be partly dependent on cellular stimulation. Since a variety of chemicals from anthropogenic, industrial, pharmaceutical, dietary, and bacterial sources bind the AhR, the ability of environmental exposures to alter AhR activity (i.e. activate or inhibit) may have a direct influence on immune function and antibody-relevant disease conditions.